Isolation of a high-affinity functional protein complex between OmcA and MtrC: Two outer membrane decaheme c-type cytochromes of Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a facultatively anaerobic bacterium capable of using soluble and insoluble forms of manganese [Mn(III/IV)] and iron [Fe(III)] as terminal electron acceptors during anaerobic respiration. To assess the structural association of two outer membrane-associated c-type decaheme cytochromes (i.e., OmcA [SO1779] and MtrC [SO1778]) and their ability to reduce soluble Fe(III)-nitrilotriacetic acid (NTA), we expressed these proteins with a C-terminal tag in wild-type S. oneidensis and a mutant deficient in these genes (i.e., Delta omcA mtrC). Endogenous MtrC copurified with tagged OmcA in wild-type Shewanella, suggesting a direct association. To further evaluate their possible interaction, both proteins were purified to near homogeneity following the independent expression of OmcA and MtrC in the Delta omcA mtrC mutant. Each purified cytochrome was confirmed to contain 10 hemes and exhibited Fe(III)-NTA reductase activity. To measure binding, MtrC was labeled with the multiuse affinity probe 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (1,2-ethanedithiol)2, which specifically associates with a tetracysteine motif engineered at the C terminus of MtrC. Upon titration with OmcA, there was a marked increase in fluorescence polarization indicating the formation of a high-affinity protein complex (Kd < 500 nM) between MtrC and OmcA whose binding was sensitive to changes in ionic strength. Following association, the OmcA-MtrC complex was observed to have enhanced Fe(III)-NTA reductase specific activity relative to either protein alone, demonstrating that OmcA and MtrC can interact directly with each other to form a stable complex that is consistent with their role in the electron transport pathway of S. oneidensis MR-1.     

Respiration of metal (hydr)oxides by Shewanella and Geobacter: a key role for multihaem c-type cytochromes

Dissimilatory reduction of metal (e.g. Fe, Mn) (hydr)oxides represents a challenge for microorganisms, as their cell envelopes are impermeable to metal (hydr)oxides that are poorly soluble in water. To overcome this physical barrier, the Gram-negative bacteria Shewanella oneidensis MR-1 and Geobacter sulfurreducens have developed electron transfer (ET) strategies that require multihaem c-type cytochromes (c-Cyts). In S. oneidensis MR-1, multihaem c-Cyts CymA and MtrA are believed to transfer electrons from the inner membrane quinone/quinol pool through the periplasm to the outer membrane. The type II secretion system of S. oneidensis MR-1 has been implicated in the reduction of metal (hydr)oxides, most likely by translocating decahaem c-Cyts MtrC and OmcA across outer membrane to the surface of bacterial cells where they form a protein complex. The extracellular MtrC and OmcA can directly reduce solid metal (hydr)oxides. Likewise, outer membrane multihaem c-Cyts OmcE and OmcS of G. sulfurreducens are suggested to transfer electrons from outer membrane to type IV pili that are hypothesized to relay the electrons to solid metal (hydr)oxides. Thus, multihaem c-Cyts play critical roles in S. oneidensis MR-1- and G. sulfurreducens-mediated dissimilatory reduction of solid metal (hydr)oxides by facilitating ET across the bacterial cell envelope.     

Extracellular polymeric substances from Shewanella sp. HRCR-1 biofilms: characterization by infrared spectroscopy and proteomics

The composition of extracellular polymeric substances (EPS) from Shewanella sp. HRCR-1 biofilms was investigated using infrared spectroscopy and proteomics to provide insight into potential ecophysiological functions and redox activity of the EPS. Both bound and loosely associated EPS were extracted from Shewanella sp. HRCR-1 biofilms prepared using a hollow-fibre membrane biofilm reactor. Fourier transform infrared spectra revealed the presence of proteins, polysaccharides, nucleic acids, membrane lipids and fatty acids in the EPS fractions. Using a global proteomic approach, a total of 58 extracellular and outer membrane proteins were identified in the EPS. These included homologues of multiple Shewanella oneidensis MR-1 proteins that potentially contribute to key physiological biofilm processes, such as biofilm-promoting protein BpfA, surface-associated serine protease, nucleotidases (CpdB and UshA), an extracellular lipase, and oligopeptidases (PtrB and a M13 family oligopeptidase lipoprotein). In addition, 20 redox proteins were found in extracted EPS. Among the detected redox proteins were the homologues of two S. oneidensis MR-1 c-type cytochromes, MtrC and OmcA, which have been implicated in extracellular electron transfer. Given their detection in the EPS of Shewanella sp. HRCR-1 biofilms, c-type cytochromes may contribute to the possible redox activity of the biofilm matrix and play important roles in extracellular electron transfer reactions.     

金属还原菌希瓦氏菌厌氧呼吸能力及其在环境修复中的研究进展

Shewanella oneidensis MR-1是一种模式金属还原菌,它能够在厌氧条件下,将多种金属化合物和人工合成染料等作为电子受体还原代谢。因此,该菌常常被用于生态修复等研究。厌氧条件下,S.oneidensis MR-1能够将细胞质内或细胞内膜产生的电子通过定位于细胞内膜、细胞膜周质和细胞外膜上的c-血红色素蛋白或还原酶所组成的具有多样性的电子传递系统,最终传递到存在于细菌细胞外环境中的电子受体。通过对多种电子传递过程的介绍,进一步阐明其对污染物修复和纳米材料合成的机理,从而为未来对该类微生物的利用和开发提供更为充分的理论依据。     

Extracellular reduction of hexavalent chromium by cytochromes MtrC and OmcA of Shewanella oneidensis MR-1

To characterize the roles of cytochromes MtrC and OmcA of Shewanella oneidensis MR-1 in Cr(VI) reduction, the effects of deleting the mtrC and/or omcA gene on Cr(VI) reduction and the cellular locations of reduced Cr(III) precipitates were investigated. Compared to the rate of reduction of Cr(VI) by the wild type (wt), the deletion of mtrC decreased the initial rate of Cr(VI) reduction by 43.5%, while the deletion of omcA or both mtrC and omcA lowered the rate by 53.4% and 68.9%, respectively. In wt cells, Cr(III) precipitates were detected by transmission electron microscopy in the extracellular matrix between the cells, in association with the outer membrane, and inside the cytoplasm. No extracellular matrix-associated Cr(III) precipitates, however, were found in the cytochrome mutant cell suspension. In mutant cells without either MtrC or OmcA, most Cr(III) precipitates were found in association with the outer membrane, while in mutant cells lacking both MtrC and OmcA, most Cr(III) precipitates were found inside the cytoplasm. Cr(III) precipitates were also detected by scanning election microscopy on the surfaces of the wt and mutants without MtrC or OmcA but not on the mutant cells lacking both MtrC and OmcA, demonstrating that the deletion of mtrC and omcA diminishes the extracellular formation of Cr(III) precipitates. Furthermore, purified MtrC and OmcA reduced Cr(VI) with apparent k(cat) values of 1.2 ± 0.2 (mean ± standard deviation) and 10.2 ± 1 s(-1) and K(m) values of 34.1 ± 4.5 and 41.3 ± 7.9 μM, respectively. Together, these results consistently demonstrate that MtrC and OmcA are the terminal reductases used by S. oneidensis MR-1 for extracellular Cr(VI) reduction where OmcA is a predominant Cr(VI) reductase.     

Cell surface exposure of the outer membrane cytochromes of Shewanella oneidensis MR-1

AIM: To determine if the outer membrane (OM) cytochromes of the metal-reducing bacterium Shewanella oneidensis MR-1 are exposed on the cell surface. METHODS AND RESULTS: MR-1 cells were incubated with proteinase K or buffer and the resulting degradation of the OM cytochromes was examined by Western blotting. The periplasmic fumarate reductase (control) was not degraded. The OM cytochromes OmcA and OmcB were significantly degraded by proteinase K (71 and 31%, respectively). Immunofluorescence confirmed a prominent cell surface exposure of OmcA and a partial exposure of OmcB and the noncytochrome OM protein MtrB. CONCLUSIONS: The cytochromes OmcA and OmcB are exposed on the outer face of the OM. SIGNIFICANCE AND IMPACT OF THE STUDY: The cell surface exposure of these cytochromes could allow them to directly contact extracellular insoluble electron acceptors (e.g. manganese oxides) and is consistent with their in vivo role.     

Exploring the biochemistry at the extracellular redox frontier of bacterial mineral Fe(III) respiration

Many species of the bacterial Shewanella genus are notable for their ability to respire in anoxic environments utilizing insoluble minerals of Fe(III) and Mn(IV) as extracellular electron acceptors. In Shewanella oneidensis, the process is dependent on the decahaem electron-transport proteins that lie at the extracellular face of the outer membrane where they can contact the insoluble mineral substrates. These extracellular proteins are charged with electrons provided by an inter-membrane electron-transfer pathway that links the extracellular face of the outer membrane with the inner cytoplasmic membrane and thereby intracellular electron sources. In the present paper, we consider the common structural features of two of these outer-membrane decahaem cytochromes, MtrC and MtrF, and bring this together with biochemical, spectroscopic and voltammetric data to identify common and distinct properties of these prototypical members of different clades of the outer-membrane decahaem cytochrome superfamily.     

Shewanella oneidensis MR-1 restores menaquinone synthesis to a menaquinone-negative mutant

The mechanisms underlying the use of insoluble electron acceptors by metal-reducing bacteria, such as Shewanella oneidensis MR-1, are currently under intensive study. Current models for shuttling electrons across the outer membrane (OM) of MR-1 include roles for OM cytochromes and the possible excretion of a redox shuttle. While MR-1 is able to release a substance that restores the ability of a menaquinone (MK)-negative mutant, CMA-1, to reduce the humic acid analog anthraquinone-2,6-disulfonate (AQDS), cross-feeding experiments conducted here showed that the substance released by MR-1 restores the growth of CMA-1 on several soluble electron acceptors. Various strains derived from MR-1 also release this substance; these include mutants lacking the OM cytochromes OmcA and OmcB and the OM protein MtrB. Even though strains lacking OmcB and MtrB cannot reduce Fe(III) or AQDS, they still release a substance that restores the ability of CMA-1 to use MK-dependent electron acceptors, including AQDS and Fe(III). Quinone analysis showed that this released substance restores MK synthesis in CMA-1. This ability to restore MK synthesis in CMA-1 explains the cross-feeding results and challenges the previous hypothesis that this substance represents a redox shuttle that facilitates metal respiration.     

Specific bonds between an iron oxide surface and outer membrane cytochromes MtrC and OmcA from Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is purported to express outer membrane cytochromes (e.g., MtrC and OmcA) that transfer electrons directly to Fe(III) in a mineral during anaerobic respiration. A prerequisite for this type of reaction would be the formation of a stable bond between a cytochrome and an iron oxide surface. Atomic force microscopy (AFM) was used to detect whether a specific bond forms between a hematite (Fe(2)O(3)) thin film, created with oxygen plasma-assisted molecular beam epitaxy, and recombinant MtrC or OmcA molecules coupled to gold substrates. Force spectra displayed a unique force signature indicative of a specific bond between each cytochrome and the hematite surface. The strength of the OmcA-hematite bond was approximately twice that of the MtrC-hematite bond, but direct binding to hematite was twice as favorable for MtrC. Reversible folding/unfolding reactions were observed for mechanically denatured MtrC molecules bound to hematite. The force measurements for the hematite-cytochrome pairs were compared to spectra collected for an iron oxide and S. oneidensis under anaerobic conditions. There is a strong correlation between the whole-cell and pure-protein force spectra, suggesting that the unique binding attributes of each cytochrome complement one another and allow both MtrC and OmcA to play a prominent role in the transfer of electrons to Fe(III) in minerals. Finally, by comparing the magnitudes of binding force for the whole-cell versus pure-protein data, we were able to estimate that a single bacterium of S. oneidensis (2 by 0.5 microm) expresses approximately 10(4) cytochromes on its outer surface.     

Characterization of Shewanella oneidensis MtrC: a cell-surface decaheme cytochrome involved in respiratory electron transport to extracellular electron acceptors

MtrC is a decaheme c-type cytochrome associated with the outer cell membrane of Fe(III)-respiring species of the Shewanella genus. It is proposed to play a role in anaerobic respiration by mediating electron transfer to extracellular mineral oxides that can serve as terminal electron acceptors. The present work presents the first spectropotentiometric and voltammetric characterization of MtrC, using protein purified from Shewanella oneidensis MR-1. Potentiometric titrations, monitored by UV–vis absorption and electron paramagnetic resonance (EPR) spectroscopy, reveal that the hemes within MtrC titrate over a broad potential range spanning between approximately +100 and approximately −500 mV (vs. the standard hydrogen electrode). Across this potential window the UV–vis absorption spectra are characteristic of low-spin c-type hemes and the EPR spectra reveal broad, complex features that suggest the presence of magnetically spin-coupled low-spin c-hemes. Non-catalytic protein film voltammetry of MtrC demonstrates reversible electrochemistry over a potential window similar to that disclosed spectroscopically. The voltammetry also allows definition of kinetic properties of MtrC in direct electron exchange with a solid electrode surface and during reduction of a model Fe(III) substrate. Taken together, the data provide quantitative information on the potential domain in which MtrC can operate.     

Current production and metal oxide reduction by Shewanella oneidensis MR-1 wild type and mutants

Shewanella oneidensis MR-1 is a gram-negative facultative anaerobe capable of utilizing a broad range of electron acceptors, including several solid substrates. S. oneidensis MR-1 can reduce Mn(IV) and Fe(III) oxides and can produce current in microbial fuel cells. The mechanisms that are employed by S. oneidensis MR-1 to execute these processes have not yet been fully elucidated. Several different S. oneidensis MR-1 deletion mutants were generated and tested for current production and metal oxide reduction. The results showed that a few key cytochromes play a role in all of the processes but that their degrees of participation in each process are very different. Overall, these data suggest a very complex picture of electron transfer to solid and soluble substrates by S. oneidensis MR-1.     

产电微生物Shewanella菌厌氧呼吸代谢网络研究进展

以模式菌株Shewanella oneidensis MR-1为代表的Shewanella菌属产电微生物广泛分布于自然水体环境中。作为兼性厌氧菌,Shewanella菌除了能进行有氧呼吸外,还能利用多种电子受体进行厌氧呼吸。通过多种细胞色素所组成的复杂电子传递网络,Shewanella菌不仅能利用渗入到周质空间的可溶性电子受体进行厌氧呼吸,更为特殊的是其能够借助电子的跨膜传递实现对胞外不溶性电子受体的异化还原代谢。本文概述了近年来Shewanella菌厌氧代谢途径的研究进展,探讨电子传递网络对Shewanella菌呼吸多样性及环境适应性的影响。     

Characterization of protein-protein interactions involved in iron reduction by Shewanella oneidensis MR-1

The interaction of proteins implicated in dissimilatory metal reduction by Shewanella oneidensis MR-1 (outer membrane [OM] proteins OmcA, MtrB, and MtrC; OM-associated protein MtrA; periplasmic protein CctA; and cytoplasmic membrane protein CymA) were characterized by protein purification, analytical ultracentrifugation, and cross-linking methods. Five of these proteins are heme proteins, OmcA (83 kDa), MtrC (75 kDa), MtrA (32 kDa), CctA (19 kDa), and CymA (21 kDa), and can be visualized after sodium dodecyl sulfate-polyacrylamide gel electrophoresis by heme staining. We show for the first time that MtrC, MtrA, and MtrB form a 198-kDa complex with a 1:1:1 stoichiometry. These proteins copurify through anion-exchange chromatography, and the purified complex has the ability to reduce multiple forms of Fe(III) and Mn(IV). Additionally, MtrA fractionates with the OM through sucrose density gradient ultracentrifugation, and MtrA comigrates with MtrB in native gels. Protein cross-linking of whole cells with 1% formaldehyde show new heme bands of 160, 151, 136, and 59 kDa. Using antibodies to detect each protein separately, heme proteins OmcA and MtrC were shown to cross-link, yielding the 160-kDa band. Consistent with copurification results, MtrB cross-links with MtrA, forming high-molecular-mass bands of approximately 151 and 136 kDa.     

Role of outer membrane c-type cytochromes MtrC and OmcA in Shewanella oneidensis MR-1 cell production, accumulation, and detachment during respiration on hematite

The iron-reducing bacterium Shewanella oneidensis MR-1 has the capacity to contribute to iron cycling over the long term by respiring on crystalline iron oxides such as hematite when poorly crystalline phases are depleted. The ability of outer membrane cytochromes OmcA and MtrC of MR-1 to bind to and transfer electrons to hematite has led to the suggestion that they function as terminal reductases when this mineral is used as a respiratory substrate. Differences in their redox behavior and hematite-binding properties, however, indicate that they play different roles in the electron transfer reaction. Here, we investigated how these differences in cytochrome behavior with respect to hematite affected biofilm development when the mineral served as terminal electron acceptor (TEA). Upon attachment to hematite, cells of the wild-type (WT) strain as well as those of a ΔomcA mutant but not those of a ΔmtrC mutant replicated and accumulated on the mineral surface. The results indicate that MtrC but not OmcA is required for growth when this mineral serves as TEA. While an OmcA deficiency did not impede cell replication and accumulation on hematite prior to achievement of a maximum surface cell density comparable to that established by WT cells, OmcA was required for efficient electron transfer and cell attachment to hematite once maximum surface cell density was achieved. OmcA may therefore play a role in overcoming barriers to electron transfer and cell attachment to hematite imposed by reductive dissolution of the mineral surface from cell respiration associated with achievement of high surface cell densities.     

Profiling the membrane proteome of Shewanella oneidensis MR-1 with new affinity labeling probes

The membrane proteome plays a critical role in electron transport processes in Shewanella oneidensis MR-1, a bacterial organism that has great potential for bioremediation. Biotinylation of intact cells with subsequent affinity-enrichment has become a useful tool for characterization of the membrane proteome. As opposed to these commonly used, water-soluble commercial reagents, we here introduce a family of hydrophobic, cell-permeable affinity probes for extensive labeling and detection of membrane proteins. When applied to S. oneidensis cells, all three new chemical probes allowed identification of a substantial proportion of membrane proteins from total cell lysate without the use of specific membrane isolation method. From a total of 410 unique proteins identified, approximately 42% are cell envelope proteins that include outer membrane, periplasmic, and inner membrane proteins. This report demonstrates the first application of this intact cell biotinylation method to S. oneidensis and presents the results of many identified proteins that are involved in metal reduction processes. As a general labeling method, all chemical probes we introduced in this study can be extended to other organisms or cell types and will help expedite the characterization of membrane proteomes.     

Anaerobic central metabolic pathways in Shewanella oneidensis MR-1 reinterpreted in the light of isotopic metabolite labeling

It has been proposed that during growth under anaerobic or oxygen-limited conditions, Shewanella oneidensis MR-1 uses the serine-isocitrate lyase pathway common to many methylotrophic anaerobes, in which formaldehyde produced from pyruvate is condensed with glycine to form serine. The serine is then transformed through hydroxypyruvate and glycerate to enter central metabolism at phosphoglycerate. To examine its use of the serine-isocitrate lyase pathway under anaerobic conditions, we grew S. oneidensis MR-1 on [1-13C]lactate as the sole carbon source, with either trimethylamine N-oxide (TMAO) or fumarate as an electron acceptor. Analysis of cellular metabolites indicated that a large percentage (>70%) of lactate was partially oxidized to either acetate or pyruvate. The 13C isotope distributions in amino acids and other key metabolites indicate that under anaerobic conditions, although glyoxylate synthesized from the isocitrate lyase reaction can be converted to glycine, a complete serine-isocitrate pathway is not present and serine/glycine is, in fact, oxidized via a highly reversible degradation pathway. The labeling data also suggest significant activity in the anapleurotic (malic enzyme and phosphoenolpyruvate carboxylase) reactions. Although the tricarboxylic acid (TCA) cycle is often observed to be incomplete in many other anaerobes (absence of 2-oxoglutarate dehydrogenase activity), isotopic labeling supports the existence of a complete TCA cycle in S. oneidensis MR-1 under certain anaerobic conditions, e.g., TMAO-reducing conditions.     

The outer membrane protein Omp35 affects the reduction of Fe(III), nitrate,and fumarate by <Emphasis Type="Italic">Shewanella oneidensis</Emphasis> MR-1

Background  

Shewanella oneidensis MR-1 uses several electron acceptors to support anaerobic respiration including insoluble species such as iron(III) and manganese(IV) oxides, and soluble species such as nitrate, fumarate, dimethylsulfoxide and many others. MR-1 has complex branched electron transport chains that include components in the cytoplasmic membrane, periplasm, and outer membrane (OM). Previous studies have implicated a role for anaerobically upregulated OM electron transport components in the use of insoluble electron acceptors, and have suggested that other OM components may also contribute to insoluble electron acceptor use. In this study, the role for an anaerobically upregulated 35-kDa OM protein (Omp35) in the use of anaerobic electron acceptors was explored.     

Hydrogenase- and outer membrane c-type cytochrome-facilitated reduction of technetium(VII) by Shewanella oneidensis MR-1

Pertechnetate, ⁹⁹Tc(VII)O₄^–, is a highly mobile radionuclide contaminant at US Department of Energy sites that can be enzymatically reduced by a range of anaerobic and facultatively anaerobic microorganisms, including Shewanella oneidensis MR-1, to poorly soluble Tc(IV)O_2(s). In other microorganisms, Tc(VII)O₄^– reduction is generally considered to be catalysed by hydrogenase. Here, we provide evidence that although the NiFe hydrogenase of MR-1 was involved in the H₂-driven reduction of Tc(VII)O₄^–[presumably through a direct coupling of H₂ oxidation and Tc(VII) reduction], the deletion of both hydrogenase genes did not completely eliminate the ability of MR-1 to reduce Tc(VII). With lactate as the electron donor, mutants lacking the outer membrane c -type cytochromes MtrC and OmcA or the proteins required for the maturation of c -type cytochromes were defective in reducing Tc(VII) to nanoparticulate TcO₂·nH₂O_(s) relative to MR-1 or a NiFe hydrogenase mutant. In addition, reduced MtrC and OmcA were oxidized by Tc(VII)O₄^–, confirming the capacity for direct electron transfer from these OMCs to TcO₄^–. c -Type cytochrome-catalysed Tc(VII) reduction could be a potentially important mechanism in environments where organic electron donor concentrations are sufficient to allow this reaction to dominate.     

Selenite and tellurite reduction by Shewanella oneidensis

Shewanella oneidensis MR-1 reduces selenite and tellurite preferentially under anaerobic conditions. The Se(0) and Te(0) deposits are located extracellularly and intracellularly, respectively. This difference in localization and the distinct effect of some inhibitors and electron acceptors on these reduction processes are taken as evidence of two independent pathways.